1. Field of the Invention
This invention relates to a protein of Brucella abortus and to a specific region thereof useful as diagnostic reagents.
2. Description of the Prior Art
Bovine brucellosis is a disease associated with abortions and infertility, and is caused by the gram-negative organism B. abortus. Despite an active vaccination program, bovine brucellosis continues to be a problem in some areas in the United States ([Hagen and Bruners' Infectious Diseases of Domestic Animals. Ithaca, NY, Gillespie, J. H., et al., eds., 7th Edition, Cornell University Press; Manthei, C. A., et al., The Yearbook of Agriculture. Proc. Anim. Dis. 84th Congr., Second Session, House Document No. 344, U.S. Department of Agriculture, 1956] and is an economically important disease. Standard serologic tests for bovine brucellosis have been in use since 1940 [Manthei et al., supra], but the most difficult task has been in distinguishing antibodies of infected from those of vaccinated animals. To date, most diagnostic tests for B. abortus rely on detecting a humoral immune response, although the most definitive diagnostic test is bacterial culture and positive identification of B. abortus [Alton, G. G., et al., Laboratory Techniques in Brucellosis. Washington, D.C., Second edition, World Health Organization, 1975]. The bovine anti-Brucella antibody response is not only directed to the lipopolysaccharide (LPS) component of the cell [Lamb, V. L., et al., Inf. Immun. 26: 240-247 (1979); Ruppanner, R., et al., Am. J. Vet. Res. 41: 1329-1332 (1980); Saunders, G. C., et al., J. Infect. Dis. 136:5258-5266 (1977), but also to the proteins and other macromolecular components [Nielsen, K. H., et al., Res. Vet. Sci. 35: 14-18 (1983); Schurig, G. G., et al., Infect. Immun. 21: 994-1002 (1978); Stemshorn, B. W., et al., Can. J. Comp. Med. 41: 152-159 (1977); Tabatabai, L. B., et al., Dev. Biol. Stand. 56: 199-211 (1984); Tabatabai, L. B., et al., Vet. Microbiol. 9: 549-560 (1984); Tabatabai, L. B., et al., J. Clin. Microbiol. 20: 209-213]. However, serologic reactions following vaccination with B. abortus abortus Strain 19 interfere with diagnosis of brucellosis (using the card test) unless supplemental tests are performed, such as the rivanol and complement fixation tests [Alton, supra; Manthei, supra]. Recent progress in the development of procedures for diagnosis of brucellosis has been reviewed [Stemshorn, B. W., Dev. Biol. Stand. 56: 325-340 (1984)]. Specifically, a competitive enzyme-linked immunosorbent assay (ELISA) procedure based on O-chain and competing monoclonal antibodies has shown promising results [Nielsen, K. H., et al., Ann. Inst. Pasteur, Microbiol. 138: 69-144 (1987)]. It has been previously reported that B. abortus salt-extractable proteins (BCSP) could be used in a sensitive ELISA procedure for detecting brucellosis in cattle [Tabatabai, L. B., et al., Vet. Microbiol., 9: 549-560 (1984)], and also for differentiating between vaccinated (calfhood vaccinated) and infected cattle using a Western blot procedure [Belzer, C. A., et al., Vet. Microbiol. 27: 12 pp. (in press, accepted for publication Sep. 20, 1990)]. However, approximately 3% of the animals that were vaccinated when sexually mature reacted also with the antigens [Belzer, supra]. We have shown that one of these proteins detects antibody to infected but not vaccinated cattle [Thompson, M., et al., Ann. Mtg. Iowa Acad. Sci., April 20-21, 1990, Drake University, Abstract No. 84]. Recently, we cloned the gene coding for this protein [Bricker, B. J., et al., Inf. Immun. 58: 2935-2939 (1990)] and have presented evidence that the recombinant protein also detects antibody to B. abortus of infected but not vaccinated animals [Thompson, supra].
As described in Tabatabai et al. [Tabatabai, L. B., et al., Infect. Immun. 26: 668-679 (1979)], and as modified in Belzer et al. (supra) washed methanol-inactivated cells of B. abortus were resuspended in M NaCl-0.1M sodium citrate and stirred overnight. The protein mixture in the supernatant thus obtained was precipitated with ammonium sulfate to obtain the protein mixture. Polyacrylamide gel electrophoresis indicated the presence of a protein of approximate molecular weight of 20,000 daltons [Tabatabai et al., Infect. Immun., supra, FIG. 4, page 674; and Tabatabai, L. B. et al., Vet. Microbiol. 20: 49-58 (1989), FIG. 1, page 55], but the procedure was not practical for use in preparing antiserum to the protein.
It has been suggested that the mixture of soluble salt-extractable proteins from B. abortus may be of potential value for preparing vaccines and/or for use as diagnostic reagents in the prevention or diagnosis of bovine brucellosis [Tabatabai et al., Vet. Microbiol. 9: 549-560 (1984), supra]. Further, the salt-extractable proteins of B. abortus have been analyzed by crossed immunoelectrophoresis using rabbit antiserum to protein antigens and by isoelectric focusing with polyacrylamide gels [Tabatabai et al., Dev. Biol. Stand., and Vet. Microbiol., supra]. The isoelectric pH's of the extracted proteins were profiled in FIG. 4, page 557, of Tabatabai et al., Vet. Microbiol., supra.